Myo-inositol 1,2,3,4,5,6-hexaphosphate, commonly known as phytic acid, is an abundant molecule in many plant seeds and vegetative tissue such as roots and tubers (Hartland and Oberlaeas, (1986) J. Assoc. Off. Anal. Chem. 69:667-670). Phytic acid exists primarily as mixture of potassium, calcium, iron, zinc and magnesium phytate salts (Pernollet J. C. (1978) Phytochemistry 17:147314 1480).
In corn (Zea mays L.), 90% of the phytate is deposited in protein bodies localized in the germ whereas in legume crops 90% of the phytate is localized in the endosperm and cotyledons. Up to 80% of phytate is in the aluerone layer of wheat (Triticum aestivum Lam.) and rice (Oryza sative L.) (O'Dell B. L. et al. (1972) J. Agric. Food Chem. 20:718-721). The presence of phytate phosphorous in such food crops decreases the bioavailability of zinc by forming a very stable insoluble phytate zink complex, making the zinc unavailable in the intestinal mucosa of mammals (O'Dell, B. L., et al. (1972) J. Agr. Food Chem. 20:718-721). Although phytate phosphorous is readily available to ruminants, it is less available to monogastric animals. In addition to being only partially digestible, the presence of phytic acid in food crops leads to excretion of other limiting nutrients such as essential amino acids, calcium and zinc (Mroz, Z. et al. (1994) J. Animal Sci. 72:126-132; Fox et al., In Nutritional Toxicology Vol. 3, Academic Press, San Diego (1989) pp. 59-96).
Phytic acid is thought to arise in plants by two pathways. The first pathway uses free myo-inositol as the initial substrate, with subsequent phosphorylation by a phosphoinositol kinase. Contribution to the free myo-inositol pool is either by recycling from other pathways or by the dephosphorylation of myo-inositol-1-phosphate. The alternate pathway uses myo-inositol-1-phosphate as the initial substrate, with subsequent phosphorylations catalyzed by phosphoinositol kinase. The committed step for myo-inositol-1-phosphate production is the NAD+-catalyzed oxidation of carbon 5 of the b-enantiomer of D-glucose-6-phosphate. This reaction is catalyzed by myo-inositol-1-phosphate synthase (Raboy, V. In Inositol Metabolism in Plants (1990) Wiley-Liss, New York, pp. 55-76).
Phytic acid is degraded in plant cells to D-myo-inositol 1,2,4,5,6-pentakisphosphate and orthophosphate through the action of phytase. Manipulation of this enzyme activity could lead to a reduction of phytic acid levels in seeds and an increase in inositol trisphosphate and free phosphate, thus making phosphorus more metabolically available to animals that are fed the seed. Another method to lower phytic acid levels is by inhibiting the activity of myo-inositol-1(or 4)-monophosphatase, which catalyzes the reaction: myo-inositol 1-phosphate+H2O=myo-inositol+orthophosphate. Manipulation of the activity of this enzyme in developing seeds could decrease phytic acid levels in seeds and increase levels of free phosphate. Lastly, phytic acid levels could also be reduced by inhibiting the activity of inositol trisphosphate kinase. This enzyme catalyzes the reaction: ATP+1D-myo-inositol 1,3,4-trisphosphate=ADP+1D-myo-inositol 1,3,4,6-tetrakisphosphate. This reaction is one of the final steps leading to the formation of Myo-Inositol 1,2,3,4,5,6-hexaphosphate (phytic acid). Reduction in the activity of the enzyme in developing seeds would interrupt phytic acid synthesis leaving the phosphate as the more metabolically available inositol trisphosphate and free phosphate.
In the United States, corn accounts for about 80% of the grain fed to all classes of livestock, including poultry, and is usually ground before feeding (Corn: Chemistry and Technology, 1987, American Association of Cereal Chemists, Inc., Edited by Stanley A. Watson and Paul E. Ramstad). A meal with decreased amounts of phytic acid and increased amounts of available phosphate would lead to improved feed efficiency in corn-containing rations, making available certain minerals especially zinc, magnesium, iron and calcium. Indeed, enzymatic treatment of soybean meal-containing rations to partially hydrolyze the phosphate groups from phytic acid improves both phosphate availability and the availability of other limiting nutrients. Also, in the wet milling of corn, phytate in the steepwater tends to precipitate, causing problems in handling, storing and transportation of the steep liquor. (Pen et al. (1993) Biotechnology 11:811-814). In light of these factors, it is apparent that corn plants with heritable, substantially reduced levels of phytic acid and increased levels of free phosphorous in their seeds would be desirable. Accordingly, the availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand carbohydrate metabolism and function in plants, provide genetic tools for the manipulation of these biosynthetic pathways, and provide a means to control carbohydrate transport and distribution in plant cells.